DNA amplification by a number of amplification methods is performed at high temperatures. For example, in PCR, repeated cycles of denaturation at 95° C., annealing around 60° C. and extension around 70° C. causes significant breakdown of the dNTP's. This may significantly affect the yield of product in later cycles. Other amplification methods such as RCA and NASBA, although isothermal, also are conducted at higher temperatures. In case of NASBA, which is performed at 41° C., the stability of nucleotides may not be very critical. However RCA may be conducted at higher temperature depending upon the polymerase used and the complexity of sequence to be amplified. Stability of nucleotides can be an issue under these conditions. It is therefore desirable to have nucleotides that can survive this repeated cycling of temperature or prolonged heating at a constant yet high temperature and hence continue to give high product yields even in later cycles of amplification and possibly cut down the number of cycles/time required to achieve desirable amplification.
The sequence of nucleotide bases in a DNA molecule can be determined in a variety of ways. The chain termination method generally involves synthesizing DNA complementary to the template strand to be sequenced by extending a primer able to hybridize to a portion of that template strand with a DNA polymerase. During the synthesis reaction, deoxynucleoside triphosphates (dNTP's) are incorporated to form a DNA fragment until a chain terminating agent, for example, a dideoxynucleoside triphosphate (ddNTP) is incorporated. Incorporation of a ddNTP prevents further DNA synthesis (a process called chain termination). The size of each DNA fragment synthesized in this procedure is then determined by gel electrophoresis and this information used to determine the sequence of nucleotides in the original template DNA. For example, Tabor and Richardson, U.S. Pat. No. 4,795,699, the entire disclosure of which is incorporated herein, describes a two step sequencing method in which an unlabeled primer is labeled in a labeling step, and then extended in the presence of excess dNTPs and a ddNTP in a chain termination step. In the labeling step, a low concentration of dNTPs is provided (one being labeled) to allow a small amount of primer extension.
In the dideoxy sequencing method, the primer may be labeled, for example with 32P, by a process using a polynucleotide kinase. Such labeling allows detection of extended primers after gel electrophoresis by auto-radiography of the resulting gel. Alternatively, a labeled dNTP may be incorporated during the process of DNA synthesis, and the presence of such labeled dNTPs detected by autoradiography or other means. To this end, the dNTP may be labeled either radioactively with 32P or 35S. In another procedure, the primer can be labeled with one or more fluorescent moieties for detection by fluorescence. In yet another procedure, the ddNTP may be labeled, for example, with a fluorescent marker.
In a sequencing reaction, the labeled dNTPs or ddNTPs partially decompose, most likely due to the thermocycling conditions, and generate labeled by-products which migrate in the separating media, thus interfering with interpretation of the true sequencing fragments. For example, labeled dNTP or ddNTP decomposition products and unreacted terminators may appear on sequencing gels or electropherogram as peaks or blobs (FIG. 1, Lanes 3 and 4, blobs result when sequencing products containing conventional terminators are directly loaded onto, an electrophoretic gel). At the present time, this problem is addressed by precipitation of the sequencing products using e.g., ethanol precipitation prior to loading (FIG. 1, lanes 1 and 2). While this reduces the contamination somewhat, the procedure is time consuming and creates a bottleneck for high throughput sequencing applications.
Thus, a process is needed for improving the clarity of sequencing data. Ideally, such a process would reduce sample preparation time and result in improved sequencing throughput. Moreover, such a method would also be economical to use. These and other concerns are addressed in greater detail below.
Recently, charge modified nucleoside-triphosphates that are either highly negatively charged so that they (or any fragmentation products) move well ahead of the sequence product fragments or highly positively charged so that they (or any fragmentation products) move in the opposite direction of the sequencing fragment when separated on a sequencing gel, have been described (WO 01/19841). These nucleotides have a string of negatively or positively charged moieties attached to the base. These nucleotides once incorporated, due to the presence of string of charges on the base, significantly affect the mobility of sequencing fragments. It is desirable to have modified nucleoside triphosphates that are either highly negatively charged or net positively charged, but after incorporation have same charge as the natural nucleotides. Therefore, mobility of the sequencing products is not affected. Even when mobility is not an issue, it is desirable to have more stable nucleoside triphosphate so that any possible complications from breakdown products are prevented.